a) Field of the Invention
This invention relates to a process for the fabrication of biosensors, especially enzyme sensors. In particular, this invention is concerned with a process for fabricating biosensors, which have a wide detection range despite of their high sensitivity, in a large quantity and with uniform quality. Sensors of such characteristics are extremely useful, for example, for the determination of blood glucose concentrations.
b) Description of the Related Art
As a glucose sensor of the above-described type, the one disclosed for example in Japanese Patent Laid-Open No. 162051/1980 has been used conventionally to determine the concentration of glucose in blood (blood glucose concentration). This glucose sensor is formed of a glucose-oxidase-immobilized film and a transducer.
The enzyme employed in the above glucose sensor, glucose oxidase converts glucose, which is contained in a solution, into gluconic acid and hydrogen peroxide by using oxygen. It is therefore possible to determine the concentration of glucose in the solution by various transducers, for example, by measuring a decrease of oxygen with an oxygen electrode, measuring an increase of hydrogen peroxide with a hydrogen peroxide electrode or by measuring an increase of gluconic acid with a pH electrode.
The concentration of oxygen dissolved in a sample solution to be determined is about 0.25 mM where the sample solution has been saturated with the surrounding air. To date, this dissolved oxygen concentration has acted as a limiting factor so that the measurable upper limit of a glucose sensor has been around 100 mg/dl. The blood glucose concentration of a diabetic may however reach as high as 500 mg/dl, so that no known glucose sensors can determine such glucose concentrations.
It is hence known to upwardly shift the measurable upper limit of a glucose sensor by using a glucose-permeation-restricted membrane which restricts permeation of glucose more than that of oxygen as disclosed, for example, in Japanese Patent Laid-Open No. 162051/1980, The Electrochemical Society Extended Abstracts, 87(2), 2276 (October, 1987) or Japanese Patent Laid-Open No. 65645/1991.
These related glucose sensors will be described specifically in detail with reference to drawings.
FIG. 5 shows in a readily-understandable manner a glucose sensor according to the technique disclosed in Japanese Patent Laid-Open No. 162051/1980. A platinum cathode 52 and a silver anode 53 are formed on an electrode support 51. A glucose-oxidase-immobilized membrane 54 and a selectively permeable membrane 55 are successively superposed on them. These platinum cathode, silver anode, glucose-oxidase-immobilized membrane and selectively permeable membrane are covered and fixed by an outer film 56 through which a hole is formed centrally. This sensor is arranged in a wall of a substitute blood vessel to determine the concentration of glucose in blood. The glucose and oxygen in the blood reach the glucose-oxidase-immobilized membrane 54 through the selectively permeable membrane 55. The glucose is oxidized by the glucose oxidase. Here, oxygen is consumed so that the concentration of dissolved oxygen drops. The extent of this drop is detected by an oxygen electrode formed of the platinum cathode 52 and the silver anode 53, thereby making it possible to determine the glucose concentration. Further, the permeation of glucose is restricted by the action of the selectively permeable membrane 55 so that the measurable upper limit of the glucose sensor increases to 500 mg/dl.
FIG. 6(A) illustrates the construction of electrodes in the glucose sensor disclosed in The Electrochemical Society Extended Abstracts, 87(2), 2276 (October, 1987), while FIG. 6(B) is a cross-sectional view of the glucose sensor. In FIG. 6(A), a platinum cathode 61 and a silver anode 63 are formed on an epoxy resin 62 and, as in the above-described sensor, function as an oxygen electrode. In FIG. 6(B), a glucose-oxidase-immobilized membrane 64 is formed over the platinum cathode 61 and a silicone rubber membrane 65 is formed over the glucose-oxidase-immobilized membrane. Upon determination of the concentration of glucose in blood, the silicone rubber membrane 65 functions as a permeation-restricted membrane to restrict permeation of the glucose, so that the measurable upper limit of the sensor is shifted upwardly. In the case of this sensor, the glucose-oxidase-immobilized membrane 64 has been formed by applying dropwise a liquid coating formulation of glucose oxidase and glutaraldehyde while the silicone rubber membrane 65 has been formed by applying dropwise a silicone emulsion.
Further, U.S. Pat. No. 5,118,404 of the present inventor also discloses an enzyme sensor equipped with a permeation-restricted membrane.
In the fabrication of each of these glucose sensors, it was possible to form the permeation-restricted membrane by directly applying a permeation-restricted membrane, which had been produced in advance, to a surface of the sensor as shown in FIG. 5 when the transducer was large. Any attempt to miniaturize the transducer, however, made it extremely difficult to apply a pre-formed permeation-restricted membrane in the above-described manner. Sensors have therefore been fabricated one by one by applying a liquid coating formulation of raw materials for the permeation-restricted membrane on each transducer and then drying the thus-applied coating formulation as illustrated in FIG. 6(A) and FIG. 6(B). The thickness and properties of each permeation-restricted membrane significantly affect the response of the resulting glucose sensor. Nevertheless, it is difficult to obtain membranes of uniform quality as long as they are formed by a coating technique. Such conventional glucose sensors therefore have widely varying characteristic values and have a low non-defective percentage. Further, they require substantial time and labor because they are fabricated one by one.
There is accordingly an outstanding demand for the development of a process for fabricating many biosensors having uniform and high response characteristics and a wide response range while including the formation of a permeation-restricted membrane in the process.